An actin mechanostat ensures hyphal tip sharpness in Phytophthora infestans to achieve host penetration

Filamentous plant pathogens apply mechanical forces to pierce their hosts surface and penetrate its tissues. Devastating Phytophthora pathogens harness a specialized form of invasive tip growth to slice through the plant surface, wielding their hypha as a microscopic knife. Slicing requires a sharp hyphal tip that is not blunted at the site of the mechanical interaction. How tip shape is controlled, however, is unknown. We uncover an actin-based mechanostat in Phytophthora infestans that controls tip sharpness during penetration. Mechanical stimulation of the hypha leads to the emergence of an aster-like actin configuration, which shows fast, local, and quantitative feedback to the local stress. We evidence that this functions as an adaptive mechanical scaffold that sharpens the invasive weapon and prevents it from blunting. The hyphal tip mechanostat enables the efficient conversion of turgor into localized invasive pressures that are required to achieve host penetration.

− 1, where Imax is the peak intensity, located at or near the tip, and Ibase the mean baseline intensity, recorded in the posterior section of the germ tube. If the F-actin concentration, judged from the LifeAct intensity, is homogeneous along the germ tube, a = 0, while strong tipaccumulation gives a >> 0.
Additional examples of this analysis are shown in Figure S4. We have confirmed that both, side (xz) and top (xy) projections give results that are qualitatively similar as shown in Figure S5.

Displacement analysis:
Our approach to measure the pathogen induced surface deformations, expressed as the displacement Δh along the surface normal, was described in detail before in Bronkhorst et al. Nature Microbiology 6, pp. 1000-1006, 2021. In short: we record three-dimensional image stacks of a fluorescent PDMS in contact with a non-fluorescent aqueous medium. To extract, for each xy-pixel, the surface location in z, denoted here as Δh, where Δh = 0 is the position of the substrate in area without deformation, we fit the intensity decay at the PDMS-medium interface to a sigmoidal function. We then perform a tilt correction, to resolve small tilt angles of the surface with respect to the imaging plane. This results in spatial (xy) maps of surface displacements (Δh), which are used in subsequent analysis.

Deformation-actin correlation analysis
We performed analyses to correlate, both spatially and temporally, the local surface displacement (Δh) with the local intensity of LifeAct-eGFP, as a F-actin marker. The code computes for each pixel the relative LifeAct-eGFP fluorescence and links this to the local displacement from the analysis described above. We then divided the data in 3 zones; adhesion zones, where Δh > +0.5µm (approximately 5x the surface displacement resolution), zones of no to weak mechanical interactions, where +0.5µm > Δh > -0.5µm, and indentation zones, where Δh < -0.5µm (approximately 5x the surface displacement resolution), and aggregated fluorescence intensity data for these different zones.

Additional data description
Cytoplasmic GFP reporter To verify that the observed increase in actin accumulation during pressure application, and prior to substrate entry, is not the result of accumulation of cytoplasm containing unbound LifeAct reporter, we performed control experiments on a P. infestans transformant that expresses a cytoplasmic GFP (Pi-14-3-GFP). We performed threedimensional confocal imaging both at higher frame rates and with lower resolution (Error! Reference source not found.A-B) and at lower frame rates with higher resolution (Error! Reference source not found.C-D). Upon contact and at the start of invasion a small increase in fluorescence is at times observed; this feature is substantially weaker as compared to the LifeAct actin reporter line Pi-LA-GFP, and does not show any microstructural features as expected. Only after host penetration, when cytoplasmic and nuclear repositioning are expected to occur to initiate the invasion process inside the host, we do observe a substantial accumulation of cytoplasm in the tip. This confirms that our reported results for the accumulation of actin in the hyphal tip prior to, and during penetration are not the result of random cytoplasmic streaming but due to mechanical feedback to the cytoskeleton.       Figure 2C&E.

a) Side view (xz maximum intensity projection) to identify the time of penetration by the formation of a protrusion of the pathogen into the host tissue, occurring at the time point 270-300s in this time sequence. b) Analysis of the local LifeAct-eGFP intensity (in the area indicated by the green circle) at the hyphal apex, showing a transient increase in LifeAct-eGFP
intensity, signaling the formation of the actin aster, whose intensity peaks 1 minute after penetration, after which the signal gradually decreases over the course of several minutes. (left, A=38min, B=43min, C=38min, D=38min, E=76min, F=37min)